Chapter
13. Investigating Translocation of Prolactin
Introduction
Analyze the Data
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Analyze the Data 13-1: Investigating Translocation of Prolactin
Imagine that you are evaluating the early steps in the translocation and processing of the secretory protein prolactin. By using an experimental approach similar to that shown in Figure 13-7, you can use truncated prolactin mRNAs to control the length of nascent prolactin polypeptides that are synthesized. When prolactin mRNA that lacks a stop codon is translated in vitro, the newly synthesized polypeptide, ending with the last codon included on the mRNA, will remain attached to the ribosome, thus allowing a polypeptide of defined length to extend from the ribosome. You have generated a set of mRNAs that encode segments of the N-terminus of prolactin of increasing length, and each mRNA can be translated in vitro by a cytosolic translation extract containing ribosomes, tRNAs, aminoacyl-tRNA synthetases, GTP, and translation initiation and elongation factors. When radiolabeled amino acids are included in the translation mixture, only the polypeptide encoded by the added mRNA will be labeled. After completion of translation, each reaction mixture is resolved by SDS-polyacrylamide gel electrophoresis, and the labeled polypeptides are identified by autoradiography.
a. The autoradiogram depicted below shows the results of an experiment in which each translation reaction was carried out either in the presence (+) or the absence (−) of microsomal membranes. Based on the gel mobility of peptides synthesized in the presence or absence of microsomes, deduce how long the prolactin nascent chain must be in order for the prolactin signal peptide to enter the ER lumen and to be cleaved by signal peptidase. (Note that microsomes carry significant quantities of signal recognition particles weakly bound to the membranes.)
Messenger RNA lengths vary by steps of 20 codons or 20 amino acids each in corresponding synthesized product. When translated in the absence or presence of microsomes, only mRNAs of 130 and 150 codons produce a product that displays any difference in size with the addition of microsomes. The 130-codon mRNA gives a product that is either full length—showing no difference in size when compared to the minus microsome product—or a smaller product that is the presumed result of signal peptidase cleavage. This suggests variable or incomplete accessibility of the product to signal peptidase. In contrast, the next-step-size-longer mRNA codes for product that is fully sensitive to signal peptidase cleavage when synthesized in the presence of microsomes. The key datum is the smaller size (faster mobility) of the product plus microsomes versus the product minus microsomes. Hence, the conclusion is that the prolactin chain must be somewhere between 130 and 150 amino acids in length for the signal sequence to be fully accessible for cleavage.
b. Given this length, what can you conclude about the conformational state(s) of the nascent prolactin polypeptide when it is cleaved by signal peptidase? The following lengths will be useful for your calculation: the prolactin signal sequence is cleaved after amino acid 31; the channel within the ribosome occupied by a nascent polypeptide is about 150 Å long; a membrane bilayer is about 50 Å thick; in polypeptides with an α-helical conformation, one residue extends 1.5 Å, whereas in fully extended polypeptides, one residue extends about 3.5 Å.
_feedback: The polypeptide must be mostly α-helical. A 100-amino-acid polypeptide as an α-helix spans 150 Å, the length of the ribosome channel. Thirty amino acids span about 50 Å, the membrane thickness. In sum, a total length of about 160 amino acids (130-amino-acid spacer) is required to space the signal peptide out by 150 Å, the necessary minimum length. Only a 60-amino-acid spacer is required to give a minimally accessible signal peptide if the polypeptide were in extended conformation.
c. In another experiment, the procedure described in part (a) was repeated, except that microsomal membranes were not present during translation, but were added after translation was complete. In this case, none of the samples shows a difference in mobility in the presence or absence of microsomes. What can you conclude about whether prolactin can be translocated into isolated microsomes posttranslationally?
_feedback: The fact that there is no prolactin cleavage if microsomal membranes are added after prolactin translation is complete indicates that prolactin must translocate cotranslationally.
d. In another experiment, each translation reaction was carried out in the presence of microsomes, and the microsomal membranes and bound ribosomes were then separated from free ribosomes and soluble proteins by centrifugation. For each translation reaction, both the total reaction (T) and the membrane fraction (M) were resolved in neighboring gel lanes. Based on the amounts of labeled polypeptide in the membrane fractions in the autoradiogram depicted below, deduce how long the prolactin nascent chain must be in order for ribosomes engaged in translation to engage the SRP and thereby become bound to microsomal membranes.
_feedback: A series of parallel reactions were done and a microsomal membrane fraction was prepared by centrifugation. No prolactin labeled polypeptide is seen in the membrane fraction for mRNA shorter than 90 codons. Hence, it is only at this nascent chain length that any engagement of ribosomes with SRP and hence binding to membrane occurs. If the mRNA is 110 codons or longer, the membrane fraction contains all labeled product found in the total reaction as indicated by the equal intensity of the gel bands. So, roughly a total chain length of 100 amino acids is required to expose the 30 amino acid signal pep-tide for SRP binding. The two bands seen with the 130-codon reaction are due to partial cleavage of the product by signal peptidase. The single band seen with the 150-codon reaction reflects full cleavage of the signal peptide by signal peptidase.
